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Daniela Di Giacomo Pascaline Gaildrat Anna Abuli Julie Abdat Thierry Frébourg Mario Tosi Alexandra Martins 《Human mutation》2013,34(11):1547-1557
Exonic variants can alter pre‐mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5′ splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers’ scores recently established by Ke et al. ( 2011 ). Comparisons of hexamer‐based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high‐throughput sequencing. 相似文献
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Houdayer C Caux-Moncoutier V Krieger S Barrois M Bonnet F Bourdon V Bronner M Buisson M Coulet F Gaildrat P Lefol C Léone M Mazoyer S Muller D Remenieras A Révillion F Rouleau E Sokolowska J Vert JP Lidereau R Soubrier F Sobol H Sevenet N Bressac-de Paillerets B Hardouin A Tosi M Sinilnikova OM Stoppa-Lyonnet D 《Human mutation》2012,33(8):1228-1238
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Background: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers to hereditary breast/ovarian cancer. However, unclassified variants (UVs) (variants with unknown clinical significance) and missense polymorphisms in BRCA1 and BRCA2 genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is still unclear. The objective of our study was to identify UVs and missense polymorphisms in Algerian breast/ovarian cancer patients and relatives tested previously for BRCA1 and BRCA2 genes germline mutations analysis.
Methods: We analyzed 101 DNA samples from 79 breast/ovarian cancer families. The approach used is based on BRCA1 and BRCA2 sequence variants screening by SSCP or High-Resolution Melting (HRM) curve analysis followed by direct sequencing. In silico analyses have been performed using different bioinformatics programs to individualize genetics variations that can disrupt the BRCA1 and BRCA2 genes function.
Results: Among 80 UVs and polymorphisms detected in BRCA1/2 genes (33 BRCA2 and 47 BRCA2), 31 were new UVs (10 BRCA2 and 21 BRCA2), 7 were rare UVs (4 BRCA2 and 3 BRCA2) and 42 were polymorphic variants (19 BRCA2 and 23 BRCA2). Moreover, 8 new missense UVs identified in this study: two BRCA1 (c.4066C>A/p.Gln1356Lys, c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2 (c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys1290Ser, c.5428G>T/p.Val1810Phe, c.6346C>G/p.His2116Asp and c.9256G>A/p.Gly3086Arg) located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded by PolyPhen2 program and could be pathogenic. In addition, 5 new BRCA2 missense UVs out of six that were found to be damaging by PolyPhen2 program, also were deleterious according to SIFT program. The rare BRCA2 UV c.5332G>A/p.Asp1778Asn was found here for the first time in co-occurrence in trans with the deleterious BRCA1 mutation c.798_799delTT/p.Ser267LysfsX19 in young breast cancer patient. Moreover, 10 new identified intronic variants with unknown clinical significance (3 BRCA1 and 7 BRCA2) in the present study, could be considered as benign, because GeneSplicer, SpliceSiteFinder and MaxEntScan prediction programs show no splice site alteration for these variants. Several missense polymorphisms of BRCA1 c.2612C>T/p.Pro871Leu, c.3548A>G/p.Lys1183Arg, c.4837A>G/p.Ser1613Gly and BRCA2 c.865A>C/p.Asn289His, c.1114A>C/p.Asn372His, c.2971A>G/p.Asn991Asp, c.7150C>A/p.Gly2384Lys have been identified with high frequency in patients who were tested negative for BRCA1 and BRCA2 mutations. These missense polymorphisms could have a role as susceptibility breast cancer markers in Algerian breast/ovarian cancer families where pathological BRCA1 and BRCA2 mutations were not present.
Conclusions: For the first time, UVs and missense polymorphisms in BRCA1 and BRCA2 genes have been identified in Algerian breast/ovarian cancer families. Evaluation of breast/ovarian cancer risk induced by the eight new missense UVs and common polymorphisms detected in our present work is on going in a larger study. 相似文献
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Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing: BRCA2 c.7976+5G > T as a case study
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Gemma Montalban Eugenia Fraile‐Bethencourt Irene López‐Perolio Pedro Pérez‐Segura Mar Infante Mercedes Durán María Concepción Alonso‐Cerezo Adrià López‐Fernández Orland Diez Miguel de la Hoya Eladio A. Velasco Sara Gutiérrez‐Enríquez 《Human mutation》2018,39(9):1155-1160
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Abdelilah Laraqui Nancy Uhrhammer Idriss Lahlou-Amine Hicham EL Rhaffouli Jamila El Baghdadi Mohamed Dehayni Rahali Driss Moussaoui Mohamed Ichou Yassir Sbitti Abderrahman Al Bouzidi Said Amzazi Yves-Jean Bignon 《International journal of medical sciences》2013,10(1):60-67
Worldwide variation in the distribution of BRCA mutations is well recognised, and for the Moroccan population no comprehensive studies about BRCA mutation spectra or frequencies have been published. We therefore performed mutation analysis of the BRCA1 gene in 121 Moroccan women diagnosed with breast cancer. All cases completed epidemiology and family history questionnaires and provided a DNA sample for BRCA testing. Mutation analysis was performed by direct DNA sequencing of all coding exons and flanking intron sequences of the BRCA1 gene. 31.6 % (6/19) of familial cases and 1 % (1/102) of early-onset sporadic (< 45 years) were found to be associated with BRCA1 mutations. The pathogenic mutations included two frame-shift mutations (c.798_799delTT, c.1016dupA), one missense mutation (c.5095C>T), and one nonsense mutation (c.4942A>T). The c.798_799delTT mutation was also observed in Algerian and Tunisian BC families, suggesting the first non-Jewish founder mutation to be described in Northern Africa. In addition, ten different unclassified variants were detected in BRCA1, none of which were predicted to affect splicing. Most unclassified variants were placed in Align-GVGD classes suggesting neutrality. c.5117G>C involves a highly conserved amino acid suggestive of interfering with function (Align-GVGD class C55), but has been observed in conjunction with a deleterious mutation in a Tunisian family. These findings reflect the genetic heterogeneity of the Moroccan population and are relevant to genetic counselling and clinical management. The role of BRCA2 in BC is also under study. 相似文献
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Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11
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Annelot Baert Eva Machackova Ilse Coene Carol Cremin Kristin Turner Cheryl Portigal‐Todd Marie Jill Asrat Jennifer Nuk Allison Mindlin Sean Young Andree MacMillan Tom Van Maerken Martin Trbusek Wendy McKinnon Marie E. Wood William D. Foulkes Marta Santamariña Miguel de la Hoya Lenka Foretova Bruce Poppe Anne Vral Toon Rosseel Kim De Leeneer Ana Vega Kathleen B. M. Claes 《Human mutation》2018,39(4):515-526
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Lovelock PK Healey S Au W Sum EY Tesoriero A Wong EM Hinson S Brinkworth R Bekessy A Diez O Izatt L Solomon E Jenkins M Renard H Hopper J Waring P Tavtigian SV Goldgar D Lindeman GJ Visvader JE Couch FJ Henderson BR Southey M Chenevix-Trench G Spurdle AB Brown MA;kConFab Investigators 《Journal of medical genetics》2006,43(1):74-83
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Variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes potentially affecting coding sequence as well as normal splicing activity have confounded predisposition testing in breast cancer. Here, we apply information theory to analyze BRCA1/2 mRNA splicing mutations categorized as VUS. The method was validated for 31 of 36 mutations known to cause missplicing in BRCA1/2 and all 26 that do not alter splicing. All single-nucleotide variants in the Breast Cancer Information Resource (BIC; Breast Cancer Information Core Database; http://research.nhgri.nih.gov/bic; last access June 1, 2010) were then analyzed. Information analysis is similar in sensitivity to other predictive methods; however, the thermodynamic basis of the theory also enables splice-site affinity to be determined accurately, which is important for assessing mutations that render natural splice sites partially functional and competition between cryptic and natural splice sites. We report 299 of 2,071 single-nucleotide BIC mutations that are predicted to significantly weaken natural sites and/or strengthen cryptic splice sites, 171 of which are not designated as splicing mutations in the database. Splicing alterations are predicted for 68 of 690 BRCA1 and 60 of 958 BRCA2 mutations designated as VUS. These analyses should be useful in prioritizing suspected mutations for downstream expression studies and for predicting aberrantly spliced isoforms generated by these mutations. 相似文献
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Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA2 Genetic Variants
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Gorka Ruiz de Garibay Alberto Acedo Zaida García‐Casado Sara Gutiérrez‐Enríquez Alicia Tosar Atocha Romero Pilar Garre Gemma Llort Mads Thomassen Orland Díez Pedro Pérez‐Segura Eduardo Díaz‐Rubio Eladio A. Velasco Trinidad Caldés Miguel de la Hoya 《Human mutation》2014,35(1):53-57
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Logan C. Walker Phillip J. Whiley Fergus J. Couch Daniel J. Farrugia Sue Healey Diana M. Eccles Feng Lin Samantha A. Butler Sheila A. Goff Bryony A. Thompson Sunil R. Lakhani Leonard M. Da Silva Sean V. Tavtigian David E. Goldgar Melissa A. Brown Amanda B. Spurdle 《Human mutation》2010,31(6):E1484-E1505
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Ane Y Steffensen Mette Dandanell Lars J?nson Bent Ejlertsen Anne-Marie Gerdes Finn C Nielsen Thomas vO Hansen 《European journal of human genetics : EJHG》2014,22(12):1362-1368
Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213−1G>A, c.670+1delG, c.4185+1G>A, and c.5075−1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302−15C>G, c.547+14delG, c.4676−20A>G, c.4987−21G>T, and c.5278−14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members. 相似文献
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Emilie Degrolard-Courcet Joanna Sokolowska Marie-Martine Padeano Séverine Guiu Myriam Bronner Carole Chery Fanny Coron C?me Lepage Caroline Chapusot Catherine Loustalot Jean-Louis Jouve Cyril Hatem Emmanuelle Ferrant Laurent Martin Charles Coutant Amandine Baurand Gérard Couillault Alexandra Delignette Salima El Chehadeh Sarab Lizard Laurent Arnould Pierre Fumoleau Patrick Callier Francine Mugneret Christophe Philippe Thierry Frebourg Philippe Jonveaux Laurence Faivre 《European journal of human genetics : EJHG》2014,22(8):979-987
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Whiley PJ Guidugli L Walker LC Healey S Thompson BA Lakhani SR Da Silva LM;kConFab Investigators Tavtigian SV Goldgar DE Brown MA Couch FJ Spurdle AB 《Human mutation》2011,32(6):678-687
Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty-six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants. 相似文献